Exercise 12.7 - Viability Cell Count

Materials

Suspension culture of cells

Sterile transfer pipettes

Stock 0.2% (w/v) Trypan blue

Hemacytometer and microscope

Procedure

Gently swirl a suspension culture to distribute the cells
evenly. Aseptically remove a small sample (0.1 ml) of cells from
the cultures. Place the sample in a separate test tube (it need
not be sterile).

Dilute 4 parts of stock Trypan Blue with 1 part of 5X saline
and add 0.1 ml of the diluted dye to your sample. Mix gently.

Set up a hemocytometer and cover slip. Immediately place a
drop of the stain/culture combination on the hemocytometer
(remember to use both sides of the hemocytometer) and wait one
minute.

Observe the cells with low power microscopy. Count the total
number of cells, and the number of stained cells.

Compute the concentration of viable cells per ml. of
culture.

Notes

Trypan Blue is a stain that is actively extruded from viable
cells, but which readily enters and stains dead cells. Therefore,
the cells which are blue are dead. The difference between the
total number of cells and the number of dead cells would be the
number of viable cells in a given aliquot of your culture. Trypan
Blue actually significantly overestimates the number of viable
cells, but is sufficient for purposes of this lab.

Approximately 30% of the cells measured as viable with Trypan
Blue will not be able to continue growth beyond a 24 hour period.